The long-term objectives of the proposed study are to characterize several putative virulence factors produced by Bacteroides gingivalis (B.g.) a species of Gram-negative bacterium implicated in the etiology of human periodontal disease, and to explore the contribution of these factors, both to general aspects of the pathogenicity of this bacterium and to its specific periodontopathic properties. Molecular genetic techniques will be employed to characterize the genes which encode these factors, as well as the protein products themselves. The virulence factors whose genes are to be cloned are proteolytic enzymes of B.g., including its collagenase, trypsin-like protease, and several arylaminopeptidases. The specific aims of the study are: (1) To clone the genes which encode these putative virulence factors. (2) To characterize the cloned genes by restriction endonuclease mapping, subcloning, insertional inactivation, maxicell analysis of expressed gene products, S1 nuclease protection studies to determine the transcription startpoint, and DNA sequencing. (3) To use the Southern hybridization procedure to survey the distribution of homologous DNA sequences in related bacterial species, and to determine the chromosomal or extrachromosomal location of the genes in the B.g. genome. (4) To introduce selectable mutations into the cloned genes using in vitro DNA mutagenesis techniques. (5) To adapt or develop a system of genetic transfer between E. coli and B.g. (6) To utilize plasmid shuttle vectors to transfer the in vitro- generated mutations into B.g. and to use plasmid incompatibility to select for the substitution of these mutations, by homologous recombination, in place of the corresponding wild-type alleles located in the genomes of the virulent parental B.g. strain. (7) To test the effects of these mutations on the overall virulence of the resulting mutant bacteria, using a guinea pig subcutaneous abcess model system. (8) To adapt the ligature-induced periodontitis model system in the cynomolgus monkey so that it can be used to test the periodontal virulence of protease-negative mutants of B.g., and to test the mutants in this system. The genes will be cloned into E. coli using a bacteriophage lambda expression vector and the desired plaques will be identified in the libraries either by the presence of the active gene products or by using specific antibody. Proteases will be detected directly on the plates containing the libraries by using a specific fluorescent substrate for bacterial collagenase, or overlays of collagen. In addition. overlays of gelatin, casein, or other non-specific substrates will be employed to detect a variety of proteases, and the cloned proteases further characterized using specific substrates, such as triple-helical collagen and the Wunsch pentapeptide (for collagenase) and BANA (for the trypsin-like protease of B.g).